Enzyme regulating ether lipid signaling pathways

Abstract

A multidimensional profiling strategy that combines activity-based proteomics and metabolomics was used to determine that an active protein, which is a previously uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and the lysophospholipids. Biochemical studies confirmed that the active protein regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of the active protein disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo.

1 . A method for treating cancer by modulating lipid signaling pathways in a cell comprising:
 a) contacting a subject with an agent that modulates the activity or expression of KIAA1363, wherein KIAA1363 comprises the amino acid sequence as set forth in SEQ ID NO:2; and   b) determining the effect of the agent on one or more of the following:
 i) cell migration, 
 ii) cell growth, 
 iii) cancer cell proliferation, 
 iv) expression of the transcription factor Fra-1, and/or 
 v) tumor growth in vivo; 
   wherein when KIAA1363 and/or at least one lysophospholipid is elevated in the cell, negatively modulating KIAA1363 activity or expression correlates with decreased cell migration, and/or cell growth, and/or decreased cancer cell proliferation, and/or decreased expression of Fra-1 associated with lipid signaling pathways and/or decreased tumor growth in vivo.   
     
     
         2 . The method of  claim 1 , wherein the lipid signaling pathway involves at least one ether lipid. 
     
     
         3 . The method of  claim 2 , further comprising determining the effect of the agent on the synthesis of PAF, wherein accumulation of PAF is associated with ether lipid signaling pathways. 
     
     
         4 . The method of  claim 1 , further comprising determining the effect of the agent on the synthesis of at least one lysophospholipid, where a decreased level of lysophospholipid is associated with ether lipid signaling pathways. 
     
     
         5 . The method of  claim 1 , wherein the agent is an inhibitor of KIAA1363 activity or negatively modulates the expression of KIAA1363 in the cell. 
     
     
         6 . The method of  claim 1 , wherein the agent is an inhibitor of KIAA1363. 
     
     
         7 . The method of  claim 6 , wherein the inhibitor is a carbamate. 
     
     
         8 . The method of  claim 2 , wherein the ether lipid is 2-acetyl monoalkylglycerol (MAGE). 
     
     
         9 . The method of  claim 3 , wherein the lysophospholipid is an alkyl-LPC or an alkyl-LPA lipid. 
     
     
         10 . The method of  claim 1 , wherein the cancer is selected from the group consisting of melanoma, breast cancer, and pancreatic cancer. 
     
     
         11 . A method of modulating lipid signaling pathways in a cell comprising:
 a) contacting the cell with an agent that modulates the activity or expression of KIAA1363, wherein KIAA1363 comprises the amino acid sequence as set forth in SEQ ID NO:2; and   b) determining the effect of the agent on
 i) the synthesis of at least one ether lipid, 
 ii) cell migration, 
 iii) cell growth 
 iv) tumor growth in vivo, 
 wherein when KIAA1363 and/or at least one ether lipid is elevated in the cell, negatively modulating KIAA1363 activity or expression correlates with a decreased level of lysophospholipid, cell migration, and/or cell growth associated with lipid signaling pathways. 
   
     
     
         12 . The method of  claim 11 , wherein the inhibitor is a carbamate having the general formula: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The method of  claim 12 , wherein the lipid signaling involves at least one ether lipid which is a 2-acetyl monoalkylglycerol (MAGE). 
     
     
         14 . The method of  claim 13 , wherein the MAGE is a C16:0 MAGE. 
     
     
         15 . The method of  claim 11 , wherein the lysophospholipid is an alkyl-LPC or an alkyl-LPA lipid. 
     
     
         16 . A method of identifying an inhibitor of KIAA1363 comprising:
 a) contacting a cell with a nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 2;   b) contacting the cells of step (a) with a first agent;   c) determining the rate of 2-acetyl monoalkylglycerol (MAGE) hydrolysis, or a derivative thereof, for each cell in the presence of the agent;   
       wherein when the rate of 2-acetyl MAGE hydrolysis decreases in the presence of the first agent, the decrease is indicative of an inhibitory effect of the first agent. 
     
     
         17 . The method of  claim 16 , further comprising transducing the cell with a second agent, wherein the second agent modulates the expression of KIAA1363. 
     
     
         18 . The method of  claim 16 , further comprising adding a second agent, wherein the first agent is a trifluoromethyl ketone (TFMK) and the second agent competes with the first agent for access to the active site of KIAA1363. 
     
     
         19 . The method of  claim 18 , wherein the second agent covalently binds to the active site. 
     
     
         20 . A method for identifying an inhibitor of KIAA1363 comprising
 a) isolating the membrane proteome from cells expressing KIAA1363;   b) contacting the membrane proteome with a thiol-reactive fluorescent reagent and 2-thioacetyl monoalkylglycerol (2-thioacetyl MAGE), in the presence and absence of a test agent under conditions which allow hydrolysis of the 2-thioacetyl MAGE by KIAA1363; and   c) determining the absorbance, wherein a decrease in the absorption in the presence of test compound as compared to the absorbance in the absence of test agent is indicative of an inhibitory effect of the test agent.   
     
     
         21 . The method of  claim 19 , wherein the cells expressing KIAA1363 are host cells transfected with nucleic acid encoding KIAA1363. 
     
     
         22 . The method of  claim 21 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         23 . The method of  claim 20 , wherein the thiol-reactive fluorescent reagent is 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). 
     
     
         24 . A method for treating cancer by modulating ether lipid signaling in a subject in need thereof, comprising: administering to said subject a therapeutically effective amount of an agent that modulates the activity or expression of KIAA1363, wherein KIAA1363 comprises the amino acid sequence as set forth in SEQ ID NO:2 and determining the effect of said agent on tumor size, tumor progression and/or life expectancy. 
     
     
         25 . The method of  claim 25 , wherein the subject is a human.